1. Field of the Invention
The present invention relates to methods of immobilizing dyes onto solid supports and films made thereby.
2. Description of the Background Art
The detection of component materials of fluids by means of test strips is of ever-increasing importance. In many cases, test strips provide a simple, economical and rapid detection means. Test strips are widely used in diagnosing urine qualitatively and semiquantitatively. Other test strips are used for detecting components in blood and serum in medical diagnosis. Furthermore, test strips are widely used for monitoring of beverages, drinking water, waste water and other fluids of industrial importance.
Of particular interest is cholesterol monitoring. Ideally, a patient checks his cholesterol daily or weekly to determine if the medication or diet should be adjusted to maintain the serum cholesterol levels within acceptable limits. However, there are no currently available assay devices which make it both simple and reliable for a lay person to monitor serum cholesterol levels.
A great number of clinical assays are currently in use which depend on enzyme-mediated decomposition of various analytes from undiluted whole blood samples. These assays are particularly convenient because they can be effected in a very short time, and, if a color could be developed with the high quantity of hydrogen peroxide released from the decomposition of the analyte, they would be greatly in demand for home and physicians' office use. Unfortunately, none of the prior art processes provides a matrix to which is bound or impregnated a dye in quantities sufficient to detect and measure quantitatively, accurately and precisely high levels of hydrogen peroxide that result from enzyme-mediated decomposition of various analytes from undiluted whole blood sample.
Peroxidase, because of it high turnover rate, good stability, ease of assay, and relatively low molecular weight has become widely used as a marker in enzyme immunoassays. There have been attempts to develop a sensitive and nonhazardous chromogenic reagent for this enzyme.
Peroxidase activity is generally monitored by the formation of a colored compound from a colorless oxygen acceptor according to the following equation: ##STR1##
Benzidine, o-tolidine, o-toluidine and o-dianisidine were commonly used oxygen acceptors. However, these compounds were shown to be carcinogenic, so that there have since been attempts to find alternative chromogens which are less hazardous.
A wide range of substrates has been described for the nonspecific peroxidase catalyzed oxidation reaction, cf., for example, U.S. Pat. Nos. 3,983,005; 4,251,629; 4,247,297; and 3,770,381. The different indicator reaction can be divided into two basic categories: (a) single chromogen, and (b) oxidative coupling of two chromogens. Commonly used chromogens such as o-dianisidine, o-tolidine, benzidine, tetramethylbenzidine (TMB) and 4-Cl-naphthol are used in dry chemistry dip stick formats, but these generally suffer from insensitivity or unstable color products.
Trinder demonstrated in 1969, in Ann. Clin. Biochem.. 6: 24-27, 1969, that the oxidative coupling of two chromogens in the presence of peroxidase gave intense colors that are relatively stable. Thus, chromogen systems such as phenol plus 4- aminoantipyrine, and N,N-dimethylaniline plus 3-methyl-2-benzothiazolinone hydrazone (MBTH) are commonly used.
A particularly convenient dry strip indicator for use in analyses in which hydrogen peroxide is generated is disclosed in Ertinghausen, U.S. Pat. No. 5,087,556, filed May 17, 1989 and U.S. Ser. No. 07/749,521, filed Aug. 26, 1991 now U.S. Pat. No. 5,234,813, which applications are hereby incorporated by reference in their entirety. In this type of strip, hydrogen peroxide is generated from the substrate by the catalytic reaction with oxidase, and the migrating sample front carrying the hydrogen peroxide oxidizes the immobilized chromogens, resulting in color development along all or part of the length of the strip. Although many chromogens oxidizable by hydrogen peroxide are known, the two biggest problems observed in using these known chromogens in such a system was that : (1) the oxidation reaction rate of the chromogens was too slow and (2) the disappearing or fading of color developed in as short as one minute after the plasma migrated past a certain zone. Chromogens which exhibited these problems included o-dianisidine, TMB, 4-chloronaphthol, 3-methyl-2-benzothiazolinone hydrazone (MBTH) plus dimethylaminobenzoic acid (DMAB), 4-methyl-naphthol, and 4-aminoantipyrine plus primaquine.
Many test strips depend upon a colored indicator dye bound to a supporting matrix, generally by covalent bonding. More particularly, the colored dye compound is formed by the coupling of a first and second component of a two component dyes system, the second component of which is covalently immobilized on the matrix. The addition of the first dye component forms a colored covalent adduct covalently immobilized to the matrix.
Nevertheless, colored dye compounds used in analytical test devices have been bound mostly unsatisfactorily to date. Typically, a colored compound is immobilized on the basis of its insolubility relative to the assay solution, which causes it to precipitate onto the matrix without covalent bonding. In other systems, generated color is absorbed, imbibed, impregnated or coated onto the supporting matrix. Patents exemplifying this approach include U.S. Pat. No. 4,069,016; U.S. Pat. No. 4,548,905; U.S. Pat. No. 4,038,031. Limited success at immobilizing color has been achieved by generating localized, precipitated color only at the surface of the solid phase through a signal generating enzyme system immobilized to the support, as disclosed in U.S. Pat. No. 4,435,504.
A number of methods of binding dyes to a matrix are well known in the art. The textile industry binds dye to textiles using mordants which, acting alone or in conjunction with a dye, become absorbed or adsorbed or otherwise intercalated and become stuck on the surface or on the fibers of the textile. Because the mordants and dyes are not covalently bound to the textile, they tend to leach out with washing or other contact with liquids, causing fading and discoloration.
Other known methods for immobilizing dye compounds include providing a dye molecule with a higher alkyl hydrophobic side chain which inserts itself into a hydrophobic substrate or support and is immobilized by hydrophilic/hydrophobic interactions, as disclosed in Bloom et al., U.S. Pat. No. 3,443,939.
Siegel et al., in European patent No. 345 460 disclose a method for covalently immobilizing colored dyes by bonding a second component of a two component dye system to a matrix. The color is formed when the first component, which may be the analyte or another dye component, covalently couples to the second component. The color formed is covalently immobilized to the matrix.
Allen et al., in U.S. Pat. No. 4,999,287, disclose a dye composition for use in a test strip comprising modified N,N-dimethylaniline, i.e., N-(omega-1,2-ethylenediamine carboxamidebutyl), N-methylaniline. This compound is coupled to a paper substrate using carbonyl diimidazole. The paper is activated by soaking the paper in 0.20 M carbonyldiimidazole in methylene chloride, followed by soaking the paper in 1.5 mg/ml of DMA derivative in methylene chloride. Following the covalent attachment of the dimethylaniline analog, the paper is soaked in a 0.5 mg/ml solution of 3-methyl-2-benzothiazolinone hydrazone (MBTH).
Cameron et al., in U.S. Pat. No. 4,394,444, disclose a test device for determination of an analyte in liquid sample comprises an analyte-responsive component comprising a pyridine nucleotide susceptible of reduction in response to the presence of said analyte, and at least one constituent interreactive with the analyte to cause reduction of the pyridine nucleotide, an uncoupler effective to generate an oxidizing substance from the reduced form of the pyridine nucleotide, a peroxidatively active substance, and an indicator which, when oxidized, is not susceptible to reduction by said reduced pyridine nucleotide. The analyte composition disclosed by Cameron et al. is generally used by adding it to a specimen such as urine, cerebrospinal fluid, tissue culture supernatant serum, plasma or whole blood. Test devices can be made by incorporating a carrier with the composition by impregnation, printing or spraying the test composition onto the carrier.
Seitz et al., in Anal. Chem. 61: 202-205, 1989, disclose the use of poly vinylalcohol as a substrate for indicator immobilization for fiber-optic chemical sensors. Glutaraldehyde and hydrochloric acid are added to a 5% (w/w) aqueous poly vinylalcohol solution. The resulting gel is clear and transparent in the visible and ultraviolet regions down to about 230 run. Cyanuric chloride is used to couple indicator to poly vinylalcohol which is then cross-linked with glutaraldehyde. The indicator is covalently bonded to the poly (vinylalcohol) prior to the crosslinking step. Dye fixation is very low (nanograms of dye per gram of poly(vinylalcohol)).
Brenner et al., U.S. Pat. No. 4,035,146, teach a standard method for bonding a compound to a substrate via the use of cyanuric chloride as a linking moiety. However, this method is merely for bonding an antimicrobial compound to a hydroxyl bearing cellulosic substrate such as bandages, clothing, undergarments and bedding, as well as leather or starch.